Abstract: White matter injury is the most common neonatal brain injury leading to poor neurologic outcomes in premature infants. This injury results in both focal and/or diffuse losses of oligodendrocytes, the myelinating cells in the brain. Hypoxia and inflammation are common and important risk factors within this vulnerable population and there are no treatment options available. A significant challenge to the development of novel treatment strategies for brain injury in neonates is the appropriate concern for safety. To de-risk innovative therapeutic development in this field, we focused on the identification of endogenous signaling molecules present in human maternal breast milk to further develop into safe and effective therapies for neonates. We identified multiple oxidized cholesterols (oxysterols) in human maternal breast milk that promote oligodendrocyte fate specification in postnatal neural stem cell populations via a sonic hedgehog-dependent mechanism. Following neonatal WMI, systemic administration of breast milk-associated oxysterol reversed the loss of periventricular oligodendrocytes and rescued associated motor deficits in our neonatal inflammatory WMI mouse model. Our long-term objective is to develop safe and effective therapy that mitigates the neurologic deficits of neonatal WMI. The objective of this proposal is to test the efficacy of 20HC therapy in a chronic hypoxia model and determine the molecular mechanism(s) of induced oligodendrogenesis. Our central hypothesis is that oxysterol therapy will improve myelination in hypoxia-induced neonatal WMI through stem cell-derived oligodendrogenesis and induced oligodendrocyte precursor cell (OPC) maturation. The rationale is that determining the efficacy of oxysterol therapy in animal models of hypoxia will lead to critical development of novel therapies to treat hypoxia-induced neonatal brain injuries. In Amis 1 & 2 this proposal we will test the efficacy of 20HC therapy in a neonatal mouse model of chronic hypoxia. Using separate genetic tools, we can determine the cellular behavior of both endogenous neural stem cells (Aim 1) as well as OPCs (Aim 2) in response to therapy. In our final Aim 3, we will explore the impact of oxysterol-induced posttranslational protein modifications on Sox10. Sox10 is a critical transcription factor that regulates oligodendrocyte maturation. Because oxysterols are found in breast milk, this approach may be further developed into a novel and safe therapeutic strategy to mitigate myelin injuries including those in the neonatal period. A comprehensive understanding of the molecular mechanisms of oxysterol-induced OPC maturation may support further testing in models of adult myelin disorders including, multiple sclerosis (MS), traumatic brain injury, and stroke.